A fast method to detect cell surface expression of thyrotropin receptor (TSHr): the microchip flow cytometry analysis

Area of Science:

• Endocrinology and Metabolism
• Molecular Biology
• Cell Biology

Background:

• Loss-of-function mutations in the thyrotropin receptor (TSHr) are associated with congenital hypothyroidism and isolated hyperthyrotropinemia.
• Accurate assessment of TSHr cell surface expression is crucial for understanding the impact of these mutations.

Purpose of the Study:

• To evaluate microchip flow cytometry as a reliable method for quantifying cell surface expression of inactivating TSHr mutations.
• To analyze the cell surface expression levels of specific TSHr mutations found in patients with isolated hyperthyrotropinemia.

Main Methods:

• Utilized microchip flow cytometry analysis on COS-7 cells transfected with various inactivating TSHr mutations (L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, M527T) and a control mutation (T477I).
• Performed binding assays and standard fluorescence-activated cell sorting (FACS) for comparative analysis.
• Measured receptor expression levels using Bmax values and fluorescence intensity.

Main Results:

• Microchip flow cytometry analysis demonstrated low cell surface expression for all tested inactivating TSHr mutants, consistent with binding assay and FACS data.
• The control mutation T477I showed significantly reduced expression compared to wild-type TSHr, validating the assay's sensitivity.
• Data from binding studies, FACS, and microchip flow cytometry showed good agreement, confirming the reliability of the microchip method.

Conclusions:

• Microchip flow cytometry is a highly reliable and efficient system for studying the cell surface expression of TSHr mutations.
• The method's requirement for a low cell number and ease of use make it favorable for clinical and research applications in endocrinology.
• Findings support the role of reduced TSHr cell surface expression in the pathophysiology of isolated hyperthyrotropinemia.